Three-dimensional Scaffold-based Systems for Primary Human Intestinal Culture Abstract The roughly 8 meters of intestine in the adult human plays essential roles in physiologic homeostasis including digestion, nutrient absorption, secretion of hormones, and immune functions. Commensurate with these essential roles, diseases of the intestine are a considerable source of human morbidity and mortality, including but not limited to inflammatory bowel diseases, infection, malabsorptive states and neoplasia. Surprisingly, despite the wealth of investigations into intestinal physiology and pathophysiology, human primary intestinal culture is essentially not utilized at all as an experimental tool. Indeed, many primary tissue types lack appropriate in vitro culture methods, and we hypothesize the dearth of appropriate three-dimensional (3D) culture technologies is the critical limitation. In response, the current proposal seeks to address this notable deficiency through the development of 3D scaffold-based systems for primary human intestinal culture within designed bioreactors. Our innovative approach combines expertise in intestinal biology with the engineering of cellular microenvironments. Through a combination of original scaffolds with tunable properties, discovery and novel application of intestinal mitogens, and theoretical and experimental design of bioreactors with controlled transport properties, we will create well-defined 3D culture systems that enable rapid, high-throughput, iterative optimization of the culture microenvironment. These systems will derive an enabling technology with transformative scientific and therapeutic applications, finally enabling in vitro studies that have never before been possible in primary culture and have instead required transformed cell lines of questionable relevance or in vivo approaches. Such novel downstream applications would include in vitro screening of pharmacologic agents modulating intestinal physiology (intestinal transport, nutrient hydrolysis, or absorption), and intestinal toxicology (chemotherapeutic agents or systemically administered therapeutics). The potential for modeling mucosal immunity (innate versus cellular/humoral) and inflammatory bowel diseases in primary human culture is present, as well as the potential modeling of neoplasia upon introduction of single exogenously introduced cancer stem cells, introduction of oncogenes, or shRNA suppression of tumor suppressors. The ex vivo expansion of intestinal tissue and/or intestinal stem cells for transplantation would be enabled by the proposed studies. Finally, the principles established by these 3D culture systems will provide a paradigmshift in the approaches available to culture diverse organs. The ability to reproducibly culture 3D tissues outside of the body will enable scientists to test new hypotheses and models of disease, to develop high-throughput screens of pharmaceutical targets, and to enable the expansion of cells for regenerative medicine therapies.